Cannabidiol Alleviates Chronic Prostatitis and Chronic Pelvic Pain Syndrome via CB2 Receptor Activation and TRPV1 Desensitization.

PURPOSE: This study elucidates the mechanism of the physiological effect of cannabidiol (CBD) by assessing its impact on lipopolysaccharide (LPS)-induced inflammation in RWPE-1 cells and prostatitis-induced by 17ß-estradiol and dihydrotestosterone in a rat model, focusing on its therapeutic potential for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). MATERIALS AND METHODS: RWPE-1 cells were stratified in vitro into three groups: (1) controls, (2) cells with LPS-induced inflammation, and (3) cells with LPS-induced inflammation and treated with CBD. Enzyme-linked immunosorbent assays and western blots were performed on cellular components and supernatants after administration of CBD. Five groups of six Sprague-Dawley male rats were assigned: (1) control, (2) CP/CPPS, (3) CP/CPPS and treated with 50 mg/kg CBD, (4) CP/CPPS and treated with 100 mg/kg CBD, and (5) CP/CPPS and treated with 150 mg/kg CBD. Prostatitis was induced through administration of 17ß-estradiol and dihydrotestosterone. After four weeks of CBD treatment, a pain index was evaluated, and prostate tissue was collected for subsequent histologic examination and western blot analysis. RESULTS: CBD demonstrated efficacy in vivo for CP/CPPS and in vitro for inflammation. It inhibited the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway by activating the CB2 receptor, reducing expression of interleukin-6, tumor necrosis factor-alpha, and cyclooxygenase-2 (COX2) (p<0.01). CBD exhibited analgesic effects by activating and desensitizing the TRPV1 receptor. CONCLUSIONS: CBD inhibits the TLR4/NF-κB pathway by activating the CB2 receptor, desensitizes the TRPV1 receptor, and decreases the release of COX2. This results in relief of inflammation and pain in patients with CP/CPPS, indicating CBD as a potential treatment for CP/CPPS.

The E3 ligase HUWE1 increases the sensitivity of CRC to oxaliplatin through TOMM20 degradation.

Continuous administration of oxaliplatin, the most widely used first-line chemotherapy drug for colorectal cancer (CRC), eventually leads to drug resistance. Increasing the sensitivity of CRC cells to oxaliplatin is a key strategy to overcome this issue. Impairment of mitochondrial function is a pivotal mechanism determining the sensitivity of CRC to oxaliplatin. We discovered an inverse correlation between Translocase of Outer Mitochondrial Membrane 20 (TOMM20) and oxaliplatin sensitivity as well as an inverse relationship between TOMM20 and HECT, UBA, and WWE domain containing E3 ligase 1 (HUWE1) expression in CRC. For the first time, we demonstrated that HUWE1 ubiquitinates TOMM20 directly and also regulates TOMM20 degradation via the PARKIN-mediated pathway. Furthermore, we showed that overexpression of HUWE1 in CRC cells has a negative effect on mitochondrial function, including the generation of ATP and maintenance of mitochondrial membrane potential, leading to increased production of ROS and apoptosis. This effect was amplified when cells were treated simultaneously with oxaliplatin. Our study conclusively shows that TOMM20 is a novel target of HUWE1. Our findings indicate that HUWE1 plays a critical role in regulating oxaliplatin sensitivity by degrading TOMM20 and inducing mitochondrial damage in CRC.

Genetic mutations affecting mitochondrial function in cancer drug resistance.

Mitochondria are organelles that serve as a central hub for physiological processes in eukaryotes, including production of ATP, regulation of calcium dependent signaling, generation of ROS, and regulation of apoptosis. Cancer cells undergo metabolic reprogramming in an effort to support their increasing requirements for cell survival, growth, and proliferation, and mitochondria have primary roles in these processes. Because of their central function in survival of cancer cells and drug resistance, mitochondria are an important target in cancer therapy and many drugs targeting mitochondria that target the TCA cycle, apoptosis, metabolic pathway, and generation of ROS have been developed. Continued use of mitochondrial-targeting drugs can lead to resistance due to development of new somatic mutations. Use of drugs is limited due to these mutations, which have been detected in mitochondrial proteins. In this review, we will focus on genetic mutations in mitochondrial target proteins and their function in induction of drug-resistance.

Identification of Survival-Specific Genes in Clear Cell Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

PURPOSE: Although mutations are associated with carcinogenesis, little is known about survival-specific genes in clear cell renal cell carcinoma (ccRCC). We developed a customized next-generation sequencing (NGS) gene panel with 156 genes. The purpose of this study was to investigate whether the survival-specific genes we found were present in Korean ccRCC patients, and their association with clinicopathological findings. MATERIALS AND METHODS: DNA was extracted from the formalin-fixed, paraffin-embedded tissue of 22 ccRCC patients. NGS was performed using our survival-specific gene panel with an Illumina MiSeq. We analyzed NGS data and the correlations between mutations and clinicopathological findings and also compared them with data from the Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) and Renal Cell Cancer-European Union (RECA-EU). RESULTS: We found a total of 100 mutations in 37 of the 156 genes (23.7%) in 22 ccRCC patients. Of the 37 mutated genes, 11 were identified as clinicopathologically significant. Six were novel survival-specific genes (ADAMTS10, CARD6, NLRP2, OBSCN, SECISBP2L, and USP40), and five were top-ranked mutated genes (AKAP9, ARID1A, BAP1, KDM5C, and SETD2). Only CARD6 was validated as an overall survival-specific gene in this Korean study (p = 0.04, r = -0.441), TCGA-KIRC cohort (p = 0.0003), RECA-EU (p = 0.0005). The 10 remaining gene mutations were associated with clinicopathological findings; disease-free survival, mortality, nuclear grade, sarcomatoid component, N-stage, sex, and tumor size. CONCLUSIONS: We discovered 11 survival-specific genes in ccRCC using data from TCGA-KIRC, RECA-EU, and Korean patients. We are the first to find a correlation between CARD6 and overall survival in ccRCC. The 11 genes, including CARD6, NLRP2, OBSCN, and USP40, could be useful diagnostic, prognostic, and therapeutic markers in ccRCC.

microRNA-200a-3p increases 5-fluorouracil resistance by regulating dual specificity phosphatase 6 expression.

Acquisition of resistance to anti-cancer drugs is a significant obstacle to effective cancer treatment. Although several efforts have been made to overcome drug resistance in cancer cells, the detailed mechanisms have not been fully elucidated. Here, we investigated whether microRNAs (miRNAs) function as pivotal regulators in the acquisition of anti-cancer drug resistance to 5-fluorouracil (5-FU). A survey using a lentivirus library containing 572 precursor miRNAs revealed that five miRNAs promoted cell survival after 5-FU treatment in human hepatocellular carcinoma Hep3B cells. Among the five different clones, the clone expressing miR-200a-3p (Hep3B-miR-200a-3p) was further characterized as a 5-FU-resistant cell line. The cell viability and growth rate of Hep3B-miR-200a-3p cells were higher than those of control cells after 5-FU treatment. Ectopic expression of a miR-200a-3p mimic increased, while inhibition of miR-200a-3p downregulated, cell viability in response to 5-FU, doxorubicin, and CDDP (cisplatin). We also showed that dual-specificity phosphatase 6 (DUSP6) is a novel target of miR-200a-3p and regulates resistance to 5-FU. Ectopic expression of DUSP6 mitigated the pro-survival effects of miR-200a-3p. Taken together, these results lead us to propose that miR-200a-3p enhances anti-cancer drug resistance by decreasing DUSP6 expression.

Decreased expression of VLDLR is inversely correlated with miR-200c in human colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers and has a high rate of morbidity and mortality worldwide. Very-low-density-lipoprotein receptor (VLDLR), a member of the low-density-lipoprotein receptor (LDLR) superfamily, is a multifunctional receptor that regulates cellular signaling by binding numerous ligands. Several studies reported the altered expression of VLDLR and suggested that VLDLR may play a critical role in tumor development by affecting cell proliferation and metastasis. However, the function of VLDLR and regulation of its expression by miRNAs have not been investigated in CRC. In the present study, we investigated the expression of VLDLR in CRC patients and found it to be significantly decreased in tumors in comparison with paired adjacent non-tumor tissues. Moreover, VLDLR over-expression inhibited the proliferation and migration of CRC cells. We also found that VLDLR expression was negatively regulated by miR-200c in CRC cells and that their expression levels were inversely correlated in CRC patients. These data suggest that VLDLR down-regulation mediated by the increased expression of miR-200c may be involved in the development of CRC.

CC2D1A and CC2D1B regulate degradation and signaling of EGFR and TLR4.

Signaling through many transmembrane receptors is terminated by their sorting to the intraluminal vesicles (ILVs) of multivescular bodies (MVBs) and subsequent lysosomal degradation. ILV formation requires the endosomal sorting complex required for transport (ESCRT) machinery. CC2D1A and CC2D1B interact with the CHMP4 family of proteins, the major subunit of the ESCRT-III complex, however, their roles in receptor degradation and signaling are poorly defined. Here, we report that CC2D1A binds to CHMP4B polymers formed on endosomes to regulate the endosomal sorting pathway. We show that depletion of CC2D1A and B accelerates degradation of EGFR and elicits rapid termination of its downstream signaling through ERK1 and 2. Depletion of CC2D1A and B promotes sorting of EGFR to ILV leading to its rapid lysosomal degradation. In addition, we show that knockdown of CC2D1A and B has similar effects on degradation and downstream signaling of another membrane receptor, TLR4. Thus, these findings suggest that CC2D1A and B may have broad effects on transmembrane receptors by preventing premature ILV sorting and termination of signaling.

MicroRNA-330-5p negatively regulates ITGA5 expression in human colorectal cancer.

Colorectal cancer (CRC), one of the most prevalent malignant cancers, has high rates pf incidence and is the fourth leading cause of cancer-related deaths for both men and women worldwide. MicroRNAs (miRNAs) play critical roles in the development of various types of cancers. miRNA­330-5p has been implicated in the progression of prostate, neuronal and pancreatic cancers by regulating proliferation, migration, invasion and epithelial-mesenchymal transition of cells. The purpose of the present study was to investigate the expression of miR-330-5p in CRC and identify its target gene(s) that may act in CRC tumorigenesis. We found that miR-330-5p expression was significantly lower in CRC tissues than that in adjacent non-tumorous tissues. Furthermore, we identified integrin α5 (ITGA5) as a new target of miR-330-5p and found that it inhibits ITGA5 expression by directly binding to the 3' untranslated region of ITGA5 mRNA. These results suggest that downregulation of miR-330-5p expression may affect CRC development via modulation of ITGA5 expression.

miR-330-5p inhibits proliferation and migration of keratinocytes by targeting Pdia3 expression.

The microRNAs (miRNAs) are a class of small non-coding RNA molecules that play important roles in cellular processes by regulating gene expression at the post-transcriptional level. In skin biology, several miRNAs have been shown to be associated with differentiation, migration and apoptosis of keratinocytes, and regulation of the hair cycle. Although the biological role of miR-330-5p has been reported in several cancers and cells, the function and molecular mechanism of miR-330-5p in skin keratinocytes have not been identified. In this study, we found that miR-330-5p inhibited the proliferation and migration of mouse keratinocytes. Among the candidate target genes of miR-330-5p searched using microarray analysis, we found that the expression of Pdia3 was directly regulated by miR-330-5p in the mouse keratinocyte. Moreover, inhibition of Pdia3 expression caused decreased proliferation and migration ability of mouse keratinocytes. Additionally, expressions of miR-330-5p and Pdia3 displayed an inverse correlation with respect to the hair cycle stage. These results indicated that regulation of Pdia3 expression by miR-330-5p is important in maintaining the hair cycle through regulation of the proliferation and migration capability of keratinocytes.

Gene-gene interaction analysis identifies a new genetic risk factor for colorectal cancer.

BACKGROUND: Adiponectin levels have been shown to be associated with colorectal cancer (CRC). Furthermore, a newly identified adiponectin receptor, T-cadherin, has been associated with plasma adiponectin levels. Therefore, we investigated the potential for a genetic association between T-cadherin and CRC risk. RESULT: We conducted a case-control study using the Korean Cancer Prevention study-II cohort, which is composed of 325 CRC patients and 977 normal individuals. Study results revealed that rs3865188 in the 5' flanking region of the T-cadherin gene (CDH13) was significantly associated with CRC (p = 0.0474). The odds ratio (OR) for the TT genotype as compared to the TA + AA genotype was 1.577 (p = 0.0144). In addition, the interaction between CDH13 and the adiponectin gene (APN) for CRC risk was investigated using a logistic regression analysis. Among six APN single nucleotide polymorphisms (rs182052, rs17366568, rs2241767, rs3821799, rs3774261, and rs6773957), an interaction with the rs3865188 was found for four (rs2241767, rs3821799, rs3774261, and rs6773957). The group with combined genotypes of TT for rs3865188 and GG for rs377426 displayed the highest risk for CRC development as compared to those with the other genotype combinations. The OR for the TT/GG genotype as compared to the AA/AA genotype was 4.108 (p = 0.004). Furthermore, the plasma adiponectin level showed a correlation with the gene-gene interaction, and the group with the highest risk for CRC had the lowest adiponectin level (median, 4.8 µg/mL for the TT/GG genotype vs.7.835 µg/mL for the AA/AA genotype, p = 0.0017). CONCLUSIONS: The present study identified a new genetic factor for CRC risk and an interaction between CDH13 and APN in CRC risk. These genetic factors may be useful for predicting CRC risk.

Identification of miR-199a-5p target genes in the skin keratinocyte and their expression in cutaneous squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules that mediate the biological cellular processes via regulation of target genes through translational repression or mRNA degradation. Among various miRNAs, miRNA-199a (miR-199a) has been known to be involved in cancer development and progression, protection of cardiomyocyte, and skeletal formation. OBJECTIVE: Although miR-199a-5p was studied in various cell types, the role of miR-199a-5p and its target genes in skin keratinocyte have not been documented. In this study, we identified target genes of miR-199a-5p in skin keratinocyte. METHODS: In order to identify the target of miR-199a-5p in keratinocyte, microarray analysis was performed. The relative expression of candidate target genes was investigated using quantitative RT-PCR and western blot analysis. To determine whether their expression was directly regulated by miR-199a-5p, luciferase reporter assay was performed. In order to investigate expression of target genes in cutaneous squamous cell carcinoma, immunohistochemistry was performed. RESULTS: We identified new target genes, Bcam, Fzd6, and Wnt7a, as well as previously known targets, Ddr1 and Podxl. We found that their expressions were directly regulated by miR-199a-5p in the skin keratinocyte using in vitro study and observed that expression of miR-199a-5p was inversely correlated with those of BCAM, FZD6 and DDR1 in the cSCC. In addition, overexpression of miR-199a-5p resulted in inhibition of the migratory capability of the skin keratinocyte. CONCLUSION: These results suggested that miR-199a-5p plays a role in pathogenesis of cSCC via inhibition of invasiveness through regulation of BCAM, FZD6 and DDR1 expression.

Hairless Up-Regulates Tgf-ß2 Expression via Down-Regulation of miR-31 in the Skin of "Hairpoor" (HrHp) Mice.

Hairless (HR) has been shown to regulate hair follicle (HF) morphogenesis and hair cycling. The Hr mutant hair loss mouse referred to as "hairpoor" (Hr(Hp)) displays overexpression of the HR protein through translational derepression. In this study, we found that 64 miRNAs were differentially expressed between the skin of Hr(Hp)/Hr(Hp) and wild type mice at P7 using miRNA-microarray analysis and miR-31 displayed the most reduced expression in Hr(Hp)/Hr(Hp) skin. In vivo observation and investigation using an in vitro reporter expression system revealed that miR-31 and pri-miR-31 were consistently down-regulated in the HR over-expressed condition. In addition, we found that the transforming growth factor ß2 (Tgf-ß2), a known catagen inducer, is the putative target of miR-31. Furthermore, Tgf-ß2 level was also increased in HR over-expressed keratinocyte and Hr(Hp)/Hr(Hp) mice. These study results suggest that HR controls Tgf-ß2 expression via regulation of miR-31, thus causing abnormal hair cycle in Hr(Hp)/Hr(Hp) mice.

Indirect modulation of sensitivity to 5-fluorouracil by microRNA-96 in human colorectal cancer cells.

5-FU is an anticancer drug that is widely used to treat cancers, including colorectal cancer (CRC); however, chemoresistance to 5-FU remains an important problem to be resolved. The role of microRNAs (miRs) in chemosensitivity has recently been studied in the development of therapeutic strategies to overcome drug resistance. Here, we focused on miR-96, which has been reported to demonstrate chemosensitivity. We investigated whether 5-FU sensitivity may be modulated by miR-96 in monolayer cells and whether this relationship also applies for drug resistance in 3D tumor spheroids (TSs). When the level of miR-96 increased, the expression of the anti-apoptotic regulator XIAP and p53 stability regulator UBE2N decreased, resulting in increased apoptosis and growth inhibition following 5-FU exposure. Transfection of miR-96 inhibitors resulted in an overexpression of XIAP and UBE2N, yet only minimal change was induced in apoptosis. Nonetheless, luciferase assay failed to show direct interactions between miR-96 and these genes. In TSs, 5-FU resistance corresponded to a significantly lower level of miR-96, however only XIAP, not UBE2N, was up-regulated demonstrating partial agreement with the 2D condition regarding target expression. Overall, these results suggest that miR-96 may modulate 5-FU sensitivity in CRC cells by promoting apoptosis; however, differential expression of target genes in TSs warrants further studies on the 5-FU resistance mechanism under 3D conditions.

A long non-coding RNA snaR contributes to 5-fluorouracil resistance in human colon cancer cells.

Several types of genetic and epigenetic regulation have been implicated in the development of drug resistance, one significant challenge for cancer therapy. Although changes in the expression of non-coding RNA are also responsible for drug resistance, the specific identities and roles of them remain to be elucidated. Long non-coding RNAs (lncRNAs) are a type of ncRNA (> 200 nt) that influence the regulation of gene expression in various ways. In this study, we aimed to identify differentially expressed lncRNAs in 5-fluorouracil-resistant colon cancer cells. Using two pairs of 5-FU-resistant cells derived from the human colon cancer cell lines SNU-C4 and SNU-C5, we analyzed the expression of 90 lncRNAs by qPCR-based profiling and found that 19 and 23 lncRNAs were differentially expressed in SNU-C4R and SNU-C5R cells, respectively. We confirmed that snaR and BACE1AS were downregulated in resistant cells. To further investigate the effects of snaR on cell growth, cell viability and cell cycle were analyzed after transfection of siRNAs targeting snaR. Down-regulation of snaR decreased cell death after 5-FU treatment, which indicates that snaR loss decreases in vitro sensitivity to 5-FU. Our results provide an important insight into the involvement of lncRNAs in 5-FU resistance in colon cancer cells.

Single nucleotide deletion mutation of KCNH2 gene is responsible for LQT syndrome in a 3-generation Korean family.

Long QT syndrome (LQTS) is characterized by the prolongation of the QT interval in ECG and manifests predisposition to life threatening arrhythmia which often leads to sudden cardiac death. We encountered a 3-generation family with 5 affected family members in which LQTS was inherited in autosomal dominant manner. The LQTS is considered an ion channel disorder in which the type and location of the genetic mutation determines to a large extent the expression of the clinical syndrome. Upon screening of the genomic sequences of cardiac potassium ion channel genes, we found a single nucleotide C deletion mutation in the exon 3 of KCNH2 gene that co-segregates with the LQTS in this family. This mutation presumably resulted in a frameshift mutation, P151fs+15X. This study added a new genetic cause to the pool of mutations that lead to defected potassium ion channels in the heart.

Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails.

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.

Neurotrophic activity of DA-9801, a mixture extract of Dioscorea japonica Thunb. and Dioscorea nipponica Makino, in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea japonica Thunb. has been traditionally used to treat polyuria and diabetes in Korea. AIM OF THE STUDY: We previously report the effects of Dioscorea japonica Thunb. extract on glucose control, NGF induction, and neuroprotection in a rodent diabetic model. Since the most potent fraction, DA-9801, was identified from a mixture of Dioscorea japonica Thunb. (DJ) and Dioscorea nipponica Makino (DN) following bioactivity-guided fractionation, here, we investigated the potential mechanism of the extract activity against diabetic peripheral neuropathy (DPN). MATERIALS AND METHODS: A 1:3 mixture of DJ and DN was extracted with ethanol (DA-9801) and further fractionated into an ethylacetate-soluble fraction (DA-9801E). Effects of these extracts on neurite outgrowth were measured in PC-12 cells and DRG neurons. Effects on cell viability and TrkA phosphorylation were evaluated in PC-12 cells. NGF induction effect was determined in primary Schwann cells as well as IMS32 cells (immortalized Schwann cells). RESULTS: No cytotoxicity was observed in PC-12 cells at the concentration below 500 µg/ml of either DA-9801 or DA-9801E. DA-9801 and DA-9801E at 100 µg/ml and 10 µg/ml, respectively, showed a significant effect on neurite outgrowth in PC-12 cells and DRG neurons in the presence of or absence a low concentration of NGF (2 ng/ml). The Trk-A phosphorylation effect of DA9801 was confirmed in PC-12 cells. An NGF induction effect of these extracts was not detected in either IMS-32 cells, or primary Schwann cells. CONCLUSIONS: The NGF agonistic activity of DA-9801 and DA-9801E was demonstrated, which may contribute to their neuroprotective effect against DPN. Studies of the detailed mechanism of these extracts as well as identification of the active components are warranted for the development of an anti-DPN drug from DJ and DN.

Effects of topically applied Korean red ginseng and its genuine constituents on atopic dermatitis-like skin lesions in NC/Nga mice.

Ginseng (the root of Panax ginseng C.A. Meyer, family Araliaceae) possesses various biological activities, including anti-inflammatory and anti-tumor actions. However, their topical effect on atopic dermatitis (AD) has not been studied yet. The aim of this study was to examine the therapeutic effects of topical Korean red ginseng saponin fraction (KRGS) and its genuine constituents on AD-like skin lesions in an AD mouse model. The therapeutic effect of topical KRGS and ginsenosides in 2-chloro-1,3,5-trinitrobenzene (TNCB)-treated NC/Nga mice was assessed by measuring the skin severity scores, ear thickness, histological changes of lesional skin including mast cell count, tissue tumor necrosis factor (TNF)-α, interleukin (IL)-4, and interferon (IFN)-γ mRNA expression, and total serum IgE. Topical administration of 0.1% KRGS, 0.1% Rh2 and 0.1% Rh2+0.1% Rg3 significantly reduced the clinical skin severity scores, ear thickness and mast cell infiltration in the TNCB-induced AD-like skin lesions. Topical application of KRGS and its constituents significantly reduced TNCB-induced increase in ear TNF-α and IL-4 mRNA expression, but not IFN-γ mRNA expression. There was little change of serum total IgE level by topical KRGS and its constituents. In this study, topical KRGS and ginsenosides Rh2 and Rg3 were found to exert an anti-inflammatory effect in vivo and proved to be beneficial in an animal model of AD. Our results suggest that KRGS and its ginsenosides Rh2 and Rg3 have potential as a topical agent for the treatment of AD.

Osmotic stress inhibits proteasome by p38 MAPK-dependent phosphorylation.

Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2.

A novel mutation in Hr causes abnormal hair follicle morphogenesis in hairpoor mouse, an animal model for Marie Unna Hereditary Hypotrichosis.

Hairpoor mice (Hr(Hp)) were derived through N-ethyl-N-nitrosourea (ENU) mutagenesis. These mice display sparse and short hair in the Hr(Hp)/+ heterozygous state and complete baldness in the Hr(Hp)/Hr(Hp) homozygous state. This phenotype was irreversible and was inherited in an autosomal semidominant manner. Hair follicles (HFs) of Hr(Hp)/+ mice underwent normal cycling and appeared normal, although smaller than those of the wild-type mice. In contrast, HFs of Hr(Hp)/Hr(Hp) mice became cyst-like structures by postnatal day (P) 21. The number and length of vibrissae decreased in a dose-dependent manner as the number of mutant alleles increased. A positional candidate gene approach was used to identify the gene responsible for the hairpoor phenotype. Genetic linkage analysis determined that the hairpoor locus is 2 cm from D14Mit34 on chromosome 14. Sequence analysis of the exons of the candidate gene hairless revealed a T-to-A transversion mutation at nucleotide position 403 (exon 2), presumably resulting in abolishment of an upstream open reading frame (uORF). In addition, we also found that the near-naked mouse (Hr(N)), a spontaneously arising mutant, harbors a A402G transition in its genome. Both mutations were in the uATG codon of the second uORF in the 5' UTR and corresponded to the mutations identified in Marie Unna Hereditary Hypotrichosis (MUHH) patients. In the present study we describe the phenotype, histological morphology, and molecular etiology of an animal model of MUHH, the hairpoor mouse.